Decapentaplegic head capsule mutations disrupt novel peripodial expression controlling the morphogenesis of the Drosophila ventral head

Drosophila adult structures derive from imaginal discs, which are sacs with apposed epithelial sheets, the disc proper (DP) and the peripodial epithelium (PE). The Drosophila TGF-beta family member decapentaplegic (dpp) contributes to the development of adult structures through expression in all imaginal discs, driven by enhancers from the 3' cis-regulatory region of the gene. In the eye/antennal disc, there is 3' directed dpp expression in both the DP and PE associated with cell proliferation and eye formation. Here, we analyze a new class of dpp cis-regulatory mutations, which specifically disrupt a previously unknown region of dpp expression, controlled by enhancers in the 5' regulatory region of the gene and limited to the PE of eye/antennal discs. These are the first described Drosophila mutations that act by solely disrupting PE gene expression. The mutants display defects in the ventral adult head and alter peripodial but not DP expression of known dpp targets. However, apoptosis is observed in the underlying DP, suggesting that this peripodial dpp signaling source supports cell survival in the DP.     

Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms

The Hedgehog (Hh) family of secreted proteins governs many developmental processes in both vertebrates and invertebrates. In Drosophila, Hh acts by blocking the formation of a truncated repressor form of Cubitus interruptus (Ci) and by stimulating the nuclear translocation and activity of full-length Ci (Ci155). In the absence of Hh, Ci155 is sequestered in the cytoplasm by forming protein complexes with Costal2 (Cos2), Fused (Fu) and Suppressor of Fused [Su(fu)]. How complex formation regulates Ci155 subcellular localization is not clear. We find that Cos2 interacts with two distinct domains of Ci155, an amino (N)-terminal domain (CDN) and a carboxyl (C)-terminal domain (CORD), and Cos2 competes with Su(fu) for binding to the N-terminal region of Ci155. We provide evidence that both N- and C-terminal Cos2 binding domains are involved in the cytoplasmic retention of Ci155 in imaginal discs. Treating imaginal discs with microtubule-destabilizing reagent nocodazole promotes nuclear translocation of Ci155, suggesting that the microtubule network plays an important role in the cytoplasmic retention of Ci155. In addition, we find that adding a nuclear localization signal (NLS) to exposed regions of Ci155 greatly facilitates its nuclear translocation, suggesting that the cytoplasmic retention of Ci155 may also depend on NLS masking.     

Cullin-3 regulates pattern formation, external sensory organ development and cell survival during Drosophila development

Ubiquitin-mediated proteolysis regulates the steady-state abundance of proteins and controls cellular homoeostasis by abrupt elimination of key effector proteins. A multienzyme system targets proteins for destruction through the covalent attachment of a multiubiquitin chain. The specificity and timing of protein ubiquitination is controlled by ubiquitin ligases, such as the Skp1-Cullin-F box protein complex. Cullins are major components of SCF complexes, and have been implicated in degradation of key regulatory molecules including Cyclin E, beta-catenin and Cubitus interruptus. Here, we describe the genetic identification and molecular characterisation of the Drosophila Cullin-3 homologue. Perturbation of Cullin-3 function has pleiotropic effects during development, including defects in external sensory organ development, pattern formation and cell growth and survival. Loss or overexpression of Cullin-3 causes an increase or decrease, respectively, in external sensory organ formation, implicating Cullin-3 function in regulating the commitment of cells to the neural fate. We also find that Cullin-3 function modulates Hedgehog signalling by regulating the stability of full-length Cubitus interruptus (Ci155). Loss of Cullin-3 function in eye discs but not other imaginal discs promotes cell-autonomous accumulation of Ci155. Conversely, overexpression of Cullin-3 results in a cell-autonomous stabilisation of Ci155 in wing, haltere and leg, but not eye, imaginal discs suggesting tissue-specific regulation of Cullin-3 function. The diverse nature of Cullin-3 phenotypes highlights the importance of targeted proteolysis during Drosophila development.     

Leg development in flies versus grasshoppers: differences in dpp expression do not lead to differences in the expression of downstream components of the leg patterning pathway

All insect legs are structurally similar, characterized by five primary segments. However, this final form is achieved in different ways. Primitively, the legs developed as direct outgrowths of the body wall, a condition retained in most insect species. In some groups, including the lineage containing the genus Drosophila, legs develop indirectly from imaginal discs. Our understanding of the molecular mechanisms regulating leg development is based largely on analysis of this derived mode of leg development in the species D. melanogaster. The current model for Drosophila leg development is divided into two phases, embryonic allocation and imaginal disc patterning, which are distinguished by interactions among the genes wingless (wg), decapentaplegic (dpp) and distalless (dll). In the allocation phase, dll is activated by wg but repressed by dpp. During imaginal disc patterning, dpp and wg cooperatively activate dll and also indirectly inhibit the nuclear localization of Extradenticle (Exd), which divide the leg into distal and proximal domains. In the grasshopper Schistocerca americana, the early expression pattern of dpp differs radically from the Drosophila pattern, suggesting that the genetic interactions that allocate the leg differ between the two species. Despite early differences in dpp expression, wg, Dll and Exd are expressed in similar patterns throughout the development of grasshopper and fly legs, suggesting that some aspects of proximodistal (P/D) patterning are evolutionarily conserved. We also detect differences in later dpp expression, which suggests that dpp likely plays a role in limb segmentation in Schistocerca, but not in Drosophila. The divergence in dpp expression is surprising given that all other comparative data on gene expression during insect leg development indicate that the molecular pathways regulating this process are conserved. However, it is consistent with the early divergence in developmental mode between fly and grasshopper limbs.     

The Drosophila segment polarity gene patched interacts with decapentaplegic in wing development.

The decapentaplegic (dpp) gene of Drosophila melanogaster encodes a polypeptide of the transforming growth factor-beta family of secreted factors. It is required for the proper development of both embryonic and adult structures, and may act as a morphogen in the embryo. In wing imaginal discs, dpp is expressed and required in a stripe of cells near the anterior-posterior compartment boundary. Here we show that viable mutations in the segment polarity genes patched (ptc) and costal-2 (cos2) cause specific alterations in dpp expression within the anterior compartment of the wing imaginal disc. The interaction between ptc and dpp is particularly interesting; both genes are expressed with similar patterns at the anterior-posterior compartment boundary of the disc, and mis-expressed in a similar way in segment polarity mutant backgrounds like ptc and cos2. This mis-expression of dpp could be correlated with some of the features of the adult mutant phenotypes. We propose that ptc controls dpp expression in the imaginal discs, and that the restricted expression of dpp near the anterior-posterior compartment boundary is essential to maintain the wild-type morphology of the wing disc.     

Dissection of the target specificity of the RNA-binding protein HOW reveals dpp mRNA as a novel HOW target

Regulation of RNA metabolism plays a major role in controlling gene expression during developmental processes. The Drosophila RNA-binding protein Held out wing (HOW), regulates an array of developmental processes in embryonic and adult growth. We have characterized the primary sequence and secondary structural requirements for the HOW response element (HRE), and show that this site is necessary and sufficient for HOW binding. Based on this analysis, we have identified the Drosophila TGFbeta homolog, dpp, as a novel direct target for HOW negative regulation in the wing imaginal disc. The binding of the repressor isoform HOW(L) to the dpp 3' untranslated region (UTR) leads to a reduction of GFP-dpp3'UTR reporter levels in S-2 cells, in an HRE site-dependent manner. Moreover, co-expression of HOW(L) in the wing imaginal disc with a dpp-GFP fusion construct led to a reduction in DPP-GFP levels in a dpp-3'UTR-dependent manner. Conversely, a reduction of the endogenous levels of HOW by targeted expression of HOW-specific double-stranded RNA led to a corresponding elevation in dpp mRNA level in the wing imaginal disc. Thus, by characterizing the RNA sequences that bind HOW, we demonstrate a novel aspect of regulation, at the mRNA level, of Drosophila DPP.     

Genetic analysis of the bone morphogenetic protein-related gene, gbb, identifies multiple requirements during Drosophila development.

We have isolated mutations in the Drosophila melanogaster gene glass bottom boat (gbb), which encodes a TGF-beta signaling molecule (formerly referred to as 60A) with highest sequence similarity to members of the bone morphogenetic protein (BMP) subgroup including vertebrate BMPs 5-8. Genetic analysis of both null and hypomorphic gbb alleles indicates that the gene is required in many developmental processes, including embryonic midgut morphogenesis, patterning of the larval cuticle, fat body morphology, and development and patterning of the imaginal discs. In the embryonic midgut, we show that gbb is required for the formation of the anterior constriction and for maintenance of the homeotic gene Antennapedia in the visceral mesoderm. In addition, we show a requirement for gbb in the anterior and posterior cells of the underlying endoderm and in the formation and extension of the gastric caecae. gbb is required in all the imaginal discs for proper disc growth and for specification of veins in the wing and of macrochaete in the notum. Significantly, some of these tissues have been shown to also require the Drosophila BMP2/4 homolog decapentaplegic (dpp), while others do not. These results indicate that signaling by both gbb and dpp may contribute to the development of some tissues, while in others, gbb may signal independently of dpp.